Methylated heparin-binding hemagglutinin recombinant mycobacterial antigen, preparation method and immunogenic compositions comprising same

ABSTRACT

The invention concerns a methylated immunogenic recombinant peptide sequence comprising mycobacterial heparin-binding hemagglutinin. The invention also concerns chemical and enzymatic methods for preparing such a sequence, the sequence being previously produced in a non-methylated recombinant form then methylated by post-translational modification. The invention further concerns recombinant tools, vectors and host cells for implementing post-translational enzymatic methylation of the recombinant HBHA. The invention finally concerns immunogenic compositions comprising methylated, native or recombinant HBHA, such compositions being useful for preparing vaccines against mycobacterial infections.

The present invention relates to the field of research and developmentof novel vaccines for the treatment of mycobacterial infections, inparticular tuberculosis.

The invention concerns a methylated immunogenic recombinant peptidesequence corresponding to heparin-binding hemagglutinin (HBHA)identified in mycobacterial strains such as Mycobacterium. tuberculosisand M bovis BCG (Menozzi et al 1996 J Exp Med 184: 993-1001).

The invention also concerns methods for preparing an immunogenic peptidesequence comprising recombinant HBHA, said sequence being methylated bypost-translational modification. In particular, the invention concernsmethods for chemical or enzymatic methylation of a peptide sequencecomprising HBHA and produced in a non methylated recombinant form.

The invention also concerns recombinant host cells, tools and vectorsfor carrying out the post-translational methylation of recombinant HBHAby chemical or enzymatic methods.

Finally, the invention concerns immunogenic compositions comprisingmethylated HBHA, native or recombinant, said compositions being used toprepare vaccines against mycobacterial infections.

Mycobacteria are bacillae with a highly diversified habitat. Dependingon the species, such bacteria can colonize the ground, water, plants,animals and/or humans. Certain species such as M smegmatis are nonpathogenic saprophytes. Other species, however, are pathogenic toanimals and/or humans to a greater or lesser extent. Thus, M aviumcauses infections in birds. M bovis is responsible for bovinetuberculosis, and has also been implicated in cases of humantuberculosis. In humans, tuberculosis is principally caused by thehighly pathogenic species M tuberculosis. M. leprae is responsible forleprosy, another human disease which is rampant in developing countries.

Currently, tuberculosis is still a major public health problem as it hasthe highest mortality for a single infectious agent. The World HealthOrganization (WHO) recorded 8.8 million cases of tuberculosis in 1995(Dolin et al 1994 Bull WHO 72: 213-220). More recently, WHO publishedalarming figures disclosing 10 million new cases of tuberculosis peryear, killing 3 million people per year (Dye et al 1999 J Am Med Assoc282: 677-686). It is estimated that one third of the world's populationis infected with M. tuberculosis. However, not every infected persondevelops the disease.

The problems raised by tuberculosis were exacerbated in the 1980s withthe emergence of the pandemic due to acquired immunodeficient syndrome(AIDS). The number of cases of tuberculosis associated withimmunodepression caused by the HIV retrovirus, responsible for AIDS, hasnot ceased to grow.

To be effective, drug treatment of tuberculosis generally has to beprolonged, especially in patients already infected with the HIV virus.In the past, M. tuberculosis infections were effectively wiped out withcertain antibiotics, including rifampicin, isoniazide and pyrasinamide.However, antibiotic therapies rapidly reached their limits in thecurative treatment of tuberculosis, firstly due to the emergence ofantibiotic-resistant strains of M. tuberculosis, in particular toisoniazide, and secondly due to the toxicity of certainanti-tuberculosis molecules, including pyrazinamide.

Only one vaccine is authorized and has been in current use for more than75 years to prevent tuberculosis infection. It is the Calmette andGuérin bacillus, known as the BCG vaccine. That vaccine consists of alive form of a strain of M. bovis isolated in 1908 from a cow andrendered avirulent in vitro to allow parenteral administration tohumans. However, that vaccine is currently the subject of controversy asit is limited, in particular as regards efficacy. According to the manyclinical trials carried out around the world, the protective efficacyobtained using the BCG vaccine is from 0 to 85% (Fine, P E, 1989 RevInfect Dis 11 Suppl 2: S353-S359). A meta-analysis suggests that themean efficacy of BCG would not exceed 50% protection against pulmonarytuberculosis (Colditz et al, 1994, Jama 271: 698-702). Further, the BCGvaccine has been shown to be relatively effective in children, while itsprotective effect is virtually zero in the adult. Further again, becausethe BCG vaccine consists of a live mycobacterial strain, itsadministration is not free from side effects on the human organism, eventhough it is an attenuated strain. Such side effects appearing afortiori in immunodeficient patients, vaccinating such patients is to beavoided. That problem cannot be overcome by killing and inactivatingBCG, because they would lose any protective effects (Orme I M, 1988,Infect. Immun 56: 3310-3312).

Thus, the present invention aims to overcome the disadvantages of theBCG vaccine by proposing a novel immunogenic composition that can beused as a vaccine against tuberculosis. This immunogenic composition canalso be used in a more general manner in the context of the preventionof mycobacterial infections.

Tuberculosis is a contact disease which is transmitted by air. Onceinhaled, M. tuberculosis germs travel to the lungs which constitute theinitial center of infection. From the lungs, the germs are rapidlydisseminated through the blood or lymphatic system to other regions ofthe organism.

The entire sequence of the genome for the current best characterized M.tuberculosis strain, namely H37Rv, has been determined and analyzed toincrease our knowledge regarding the biology of this pathogen and toidentify new targets that could be used to develop novel therapeutictreatments, i.e., prophylactic or curative treatments (Cole et al, 1998,Nature 393: 537-544). The current approach consists of creating genomiclibraries from the DNA of M. tuberculosis and screening those librariesto identify novel potential therapeutic targets. Interestingly, it hasbeen observed that M. tuberculosis strains exhibit a high genetichomogeneity, the nucleotide changes from one sequence to another beingvery rare. Further, the majority of the proteins are identical acrossthe strains of this species. This is particularly important as regardsimmunity and the development of vaccines, as the antigenic markers to bescreened are almost ubiquitous.

Despite the high incidence of mycobacterial infections, little is knownabout the primary molecular mechanisms involved in their pathogenesis.

One of the major events in the pathogenesis of tuberculosis is theadhesion of microorganisms to target cells. Alveolar macrophages havelong been considered to be the portal of entry for M. tuberculosis andare assumed to transport the bacteria from the lungs to the otherorgans. However, it has recently been shown that M. tuberculosis is alsoable of interacting with epithelial cells, including M cells, whichcould allow the bacillus to directly cross the epithelial barrier(Teitelbaum et al, 1999 Immunity 10: 641-650). The relative contributionof each of these mechanisms as well as the bacterial factors involved inextra-pulmonary dissemination of M. tuberculosis is still unknown.

M. tuberculosis strains produce an adhesin termed HBHA (heparin-bindinghemagglutinin adhesion) on their surface (Menozzi et al, 1996 J Exp Med184: 993-1001). That protein is also produced by other pathogenicmycobacteria, such as M. leprae and M. avium (Reddy et al, 2000 J InfectDis 181: 1189-1193). In contrast, HBHA is not produced by the nonpathogenic saprophyte species M. smegmatis (Pethe et al, 2001 MolMicrobiol 39: 89-99).

Binding of M. tuberculosis to epithelial cells is inhibited by anti-HBHAantibodies or by competition with heparin. This is not the case withmacrophages, and so that observation suggests that the adhesionconferred by HBHA is specific to non phagocyte cells. The mechanism forthis adhesion relies on recognition, by the lysine-rich carboxy terminaldomain of HBHA (Pethe et al, 2000 J Biol Chem 275: 14273-14280), ofreceptors containing sulphated glycosaminoglycans carried by theepithelial cells.

More recently, studies have shown that HBHA plays neither a preponderantrole in the initial steps of tuberculosis infection, nor in thepersistence of mycobacteria in the lungs (Pethe et al, 2001 Nature 412:190-194). That team also showed that HBHA was not required forcolonization and survival in the spleen. In contrast, HBHA plays acrucial role in extra-pulmonary mycobacterial dissemination.Consequently, that adhesin is a virulence factor, the binding of whichto non-phagocytary cells represents an essential step in thedissemination of mycobacteria from the lungs to the spleen andpotentially to other organs such as the liver, bones, the kidneys or,possibly, the brain.

The present invention is aimed at using the antigenic power of HBHAwithin the context of an essentially prophylactic treatment, the role ofHBHA being of primary importance in the dissemination of microorganismsin infected subjects.

The cloning of the gene encoding HBHA and its expression in Escherichiacoli have suggested that the protein undergoes post-translationalmodification (Menozzi et al, 1998 Proc Natl Acad Sci USA, 95:12625-12630). In that publication, the authors hypothesized that nativeHBHA could be glycosylated, which hypothesis was subsequently shown tobe inexact. More recent work has shown that the only covalentpost-translational modification undergone by the HBHA produced by M.tuberculosis is a complex methylation of lysine residues contained inthe carboxy-terminal domain of the protein.

Within the context of the present invention, the inventors show thenature of the post-translational modification carried by the nativeHBHA, namely a complex covalent methylation, said modification endowingit with a protective antigenic power against mycobacterial infections.The peptide sequence of the recombinant HBHA produced after expressingits gene in E. coli, for example, exhibits no protective activity, as itdoes not undergo post-translational modification, like native HBHA.

Thus, the invention concerns an immunogenic recombinant peptide sequencecomprising a methylated antigen corresponding to native HBHA or to theC-terminal portion thereof.

Within the context of the present invention, the term “peptide sequence”designates all or a portion of the sequence for the HBHA protein,provided that said “peptide sequence” contains at least the lysine-richcarboxy-terminal region which ensures heparin binding. The sequence forsaid carboxy-terminal region is as follows:

KKAAPAKKAAPAKKAAPAKKAAAKKAPAKKAAAKKVTQK

This sequence was disclosed in the International patent publication withpublication number WO 97/44463.

The term “protein”, “HBHA protein” or “HBHA” as used in the presentinvention means all or a portion of the peptide sequence for HBHA,provided that it includes at least the C-terminal region of said HBHA.When the sequence under consideration comprises at most the C-terminalregion of the HBHA, the term “peptide” will advantageously be used. Theterm “peptides” will be used to designate products from the enzymaticdigestion of HBHA. However, the use of the term “peptide” is not limitedto this instance, “peptide” also being synonymous with “protein” withinthe context of the invention.

A “recombinant” peptide sequence in accordance with the inventioncorresponds to a peptide sequence obtained by expression, in aheterologous cell host, of a nucleotide sequence encoding said peptidesequence. In particular, said heterologous cell host can be a bacteriumthat does not belong to the Mycobacterium genus, for example E. coli, orother organisms such as yeasts or animal or plant cells.

The expression “nucleotide sequence” designates any DNA sequenceencoding a peptide sequence as defined in the context of the presentinvention.

In accordance with accepted use, an “antigen” designates any peptidesequence of the present invention having an immunogenic power. Inparticular, an antigen of the invention could be restricted to thecarboxy-terminal heparin binding region of HBHA.

Within the context of the invention, the expressions “heparin-bindingcarboxy-terminal region”, “heparin-binding region”, “carboxy-terminalregion” and “C-terminal region” of HBHA designates the same region ofsaid HBHA, the sequence for which is given above. Thus, theseexpressions are equivalent.

Preferably, the immunogenic recombinant peptide sequence of the presentinvention is methylated at the heparin-binding region of the HBHA. Inparticular, the methyl groups are carried by lysine residues present insaid heparin-binding region.

In a more preferred embodiment of the present invention, the methylgroups are carried by all or only part of the lysine residues present inthe C-terminal region of HBHA, provided that the methylated peptidesequence has an immunogenic activity.

Advantageously, at least thirteen lysine residues out of the fifteenpresent in the C-terminal region are methylated. The two non-methylatedlysine residues are the amino acids distal to the sequence indicatedabove for the C-terminal region of the HBHA.

The methylated lysine residues are preferably mono- or di-methylated.

In the publication by Menozzi et al, 1998, supra, it was also shown thatnative HBHA was recognized by two monoclonal antibodies, namely 3921E4and 4057D2 (Rouse et al, 1991 Infect Immun 59: 2595-2600), while therecombinant form of HBHA not post-translationally modified was notrecognized by antibody 4057D2, indicating that one of the epitopes ofnative HBHA was absent from recombinant HBHA.

The immunogenic recombinant peptide sequence of the present invention,namely the recombinant form of HBHA methylated in a post-translationalmanner, is recognized by the monoclonal antibody 4057D2, in contrast tothe non methylated recombinant form of said HBHA, as will be describedin the examples below.

The invention also concerns methods for preparing an immunogenic peptidesequence comprising recombinant HBHA, said sequence being methylated bypost-translational modification.

In particular, a preparation method of the present invention comprisesat least the following steps:

-   -   a) producing the recombinant HBHA protein in a heterologous host        cell—this form of HBHA being non methylated;    -   b) purifying said protein using conventional methods; and    -   c) post-translational methylation of the purified recombinant        HBHA.

It is understood that in the context of the invention, the HBHA proteinpurification step can be carried out before or, in another embodiment,after the protein methylation step.

The preparation method of the invention can produce methylatedrecombinant HBHA protein or, alternatively, any methylated peptidecomprising at least the heparin-binding region of said protein. Inparticular, said methylated peptide obtained by the method of theinvention corresponds to said heparin-binding region of the HBHA.

Advantageously, the heterologous host cell used in the preparationmethod of the invention is a bacterium, in particular E. coli or M.smegmatis. In particular, the host used is M. smegmatis.

Protein purification methods are known to the skilled person and do notform part of the present invention per se. As an example, theheparin-binding properties conferred by the C-terminal region of HBHAcan be exploited by purifying said HBHA by affinity on aheparin-sepharose column (Pethe et al, 2000, supra).

In particular, the invention concerns methods for chemical and enzymaticmethylation of a peptide sequence comprising HBHA previously produced ina non methylated recombinant form.

The term “production in a recombinant form” means producing a peptide byexpression in any heterologous prokaryotic or eukaryotic host.Production can be carried out from a cell culture or in vivo, such as inmilk or in a plant.

The chemical methylation of the invention is derived from the literature(Means G E, 1977 Meth Enzymol 47: 469-478). In particular, the chemicalmethylation reaction is carried out in a solution comprisingformaldehyde and NaBH₄.

The enzymatic methylation methods of the invention can be carried outusing one or more mycobacterial methyltransferases. Saidmethyltransferases catalyze the transfer of methyl groups from a donorto an acceptor, in this instance the peptide sequence for the previouslypurified recombinant HBHA. The methyl radical donor can beS-adenosylmethionine (AdoMet), which is well known to the skilledperson.

More particularly, the methyltransferase or methyltransferases arepresent and active in extracts from total mycobacterial proteins such asM. bovis BCG or M. smegmatis.

In a further embodiment of the present invention, the mycobacterialmethyltransferase or methyltransferases are purified from total proteinextracts from mycobacterial strains, before being placed in the reactionmedium to catalyze the transmethylation reaction or reactions from thedonor to the acceptor.

The invention also concerns recombinant host cells, vectors and toolsfor carrying out the enzymatic post-translational methylation ofrecombinant HBHA.

In particular, the invention concerns a recombinant host cell that canco-express nucleotide sequences encoding HBHA and mycobacterialmethyltransferase(s). Said host cell is preferably a bacteria, inparticular a strain of E. coli.

The term “co-express” as used in the present invention means the facultyof a given host cell to express at least two distinct nucleotidesequences.

In one embodiment of the present invention, the host cell ischaracterized in that it simultaneous holds at least two recombinantvectors, one of which encodes HBHA while the other(s) encode themycobacterial methyltransferase(s).

In particular, the host cell of the invention holds as many recombinantvectors as there are different proteins to be produced, each vector thenencoding a distinct recombinant mycobacterial protein.

The terms “vector”, “expression vector” and “plasmid” are used in thecontext of the present invention to designate the same cloning tool andexpression of nucleotide sequences in a manner that is conventional forthe skilled person.

In a further embodiment of the invention, all of the recombinantmycobacterial proteins or only a part thereof are encoded by the sameexpression vector.

In particular, the host cell holds a single expression vector from whichall of the mycobacterial proteins are produced, namely HBHA and themethyltransferase or methyltransferases.

When the host cell holds a single vector, the production of eachmycobacterial protein, HBHA or methyltransferase, is controlled bydistinct regulation sequences or, in a further embodiment, by the sameregulation sequences.

In particular, the production of all or a part of the recombinantproteins is controlled by the same regulation sequences.

An expression vector of the present invention advantageously encodesHBHA and at least one mycobacterial methyltransferase.

Alternatively, an expression vector of the invention encodes a singlerecombinant mycobacterial protein selected from HBHA and themethyltransferase or methyltransferases.

The present invention concerns not only the host cells and theexpression vectors as defined above considered per se, but alsoimplementation of the enzymatic methylation methods of the invention.

The present invention also pertains to a method for producing animmunogenic peptide sequence comprising recombinant HBHA, said sequencebeing methylated by post-translational modification, said methodcomprising at least the following steps:

-   -   a) co-producing the HBHA protein and the mycobacterial        methyltransferase or methyltransferases by a host cell as        defined above;    -   b) post-translational methylation of the recombinant HBHA by the        recombinant methyltransferase or methyltransferases; and    -   c) purifying the methylated recombinant HBHA using conventional        methods.

The invention also concerns methylated immunogenic recombinant peptidesequences that can be obtained in vivo using an enzymatic method or invitro using a chemical or enzymatic method.

Finally, the invention concerns immunogenic compositions comprisingmethylated HBHA, native or recombinant, said compositions being used toprepare vaccines against mycobacterial infections.

In particular, an immunogenic composition of the present inventioncomprises, in a pharmaceutically acceptable formulation, an activeprinciple which is a methylated peptide sequence selected from thepeptide sequence for native HBHA and the peptide sequence forrecombinant HBHA.

A “pharmaceutically acceptable formulation” as used in the presentinvention corresponds to a drug formulation that can be used in humansin acceptable in vivo doses having regard to the toxicity andpharmacology of the compounds concerned, while being effective on atherapeutic level, in particular on an immunogenic level.

In a preferred embodiment of the invention, the methylated peptidesequence acting as the active principle is associated with one or moreadjuvants.

The term “adjuvant” or “adjuvant compound” as used in the presentinvention means a compound that can induce or increase the specificimmune response towards an antigen or immunogen, said responseconsisting of a humoral and/or cellular response. Said immune responsegenerally occurs via stimulation of the synthesis of specificimmunoglobulins for a given antigen, in particular IgG, IgA and IgM, orof cytokines.

The active principle, methylated HBHA peptide sequence, as well as theadjuvant or adjuvants are generally mixed with pharmaceuticallyacceptable excipients such as water, a saline buffer, dextrose,glycerol, ethanol, or mixtures thereof.

Said immunogenic compositions are prepared in the form of liquidsolutions or injectable suspensions or in the solid form, for examplefreeze dried, suitable for dissolution prior to injection.

An immunogenic composition of the present invention is formulated toallow administration by diverse routes such as nasally, orally,sub-cutaneously, intradermally, intramuscularly, vaginally, rectally,ocular, or auricular. In particular, the choice of auxiliary compoundsis dictated by the selected mode of administration. Said auxiliarycompounds can in particular be wetting agents, emulsifying agents orbuffers.

Advantageously, an immunogenic composition of the invention comprises,per dose, 0.1 to 20 μg, preferably 5 μg of purified HBHA protein.

The present invention is illustrated in a non-limiting manner in theaccompanying figures in which:

FIG. 1 shows the determination of the mass of the peptide correspondingto the heparin-binding region of native and recombinant HBHA. Said HBHAswere digested overnight with Endoproteinase Glu-C (Endo-GLu;EC3.4.24.33). The fragments corresponding to the heparin-binding regionwere purified by HPLC. The fragment weight of recombinant HBHA (A) andnative HBHA (B) were then analyzed by mass spectroscopy;

FIG. 2: shows the heparin-binding region of HBHA produced by M. bovisBCG or M smegmatis (methylated recombinant HBHA). The lysines modifiedto mono- or di-methyllysines were identified using the Edman degradationtechnique;

FIG. 3: determination of the weight of the peptide corresponding to theheparin-binding region of non methylated recombinant HBHA and ofchemically methylated recombinant HBHA. The different forms of HBHAunderwent digestion with Endo-Glu overnight. The fragments correspondingto the heparin-binding region were purified by HPLC. The weight offragments of non methylated recombinant HBHA (A), recombinant HBHAchemically methylated for 6 min (B), 31 min (C) and 120 min (D), wereanalyzed by mass spectrometry.

FIG. 4: SDS-PAGE and immunoblot analysis of recombinant HBHA (1),recombinant HBHA chemically methylated for 6 min (2), 31 min (3), 120min (4) and native HBHA (5). The immunoblot analyses were carried outusing two monoclonal antibodies 3921E4 and 4057D2 (Rouse et al, 1991,supra).

FIG. 5: measure of immune cell response induced by injecting differentpreparations. Spleen cells from four mice per group were placed inculture ten weeks after the initial immunization. The cells wereunstimulated (NS) or stimulated (S) for 72 h with native HBHA (2 μg/ml).The concentration of IFN-γ was then assayed in the culture supernatants.

The invention will be better understood from the following detaileddescription which is given purely by way of illustration. It should beunderstood that the present invention is not in any way limited toexamples figuring in the detailed description.

DETAILED DESCRIPTION OF THE INVENTION

I—Materials and Methods

I-1—Bacterial Strains and Culture Conditions

Strains of M bovis BCG 1173P2 (OMS), M. tuberculosis MT103 and M.smegmatis MC²155 were cultivated in Sauton medium (Menozzi et al, 1996,supra). The E. coli BL21(DE3)pET-hbhA) strain (Pethe et al, 2000, supra)was cultivated in LB medium supplemented with 30 μg/ml of kanamycin.

I-2—Purification of HBHA

Native and recombinant HBHA were isolated as described (Menozzi et al,1996, supra; Pethe et al, 2000, supra). The final purification step wascarried out using reverse phase HPLC (Beckman Gold system) using anucleosyl-C18 type column equilibrated in 0.05% trifluoroacetic acid.Elution was carried out using a linear gradient of 0 to 80% acetonitrileprepared in 0.05% trifluoroacetic acid.

I-3—Analysis of Peptides or Proteins by Mass Spectrometry

The samples (0.1 to 10 picomoles) were prepared by the “dry drop”method.

For peptides, a 0.5 μl volume of solution was mixed withα-cyano-4-hydroxycinnamic acid extemporaneously dissolved in an amountof 10 mg/ml in a solution containing 50% CH₃CN and 0.1% trifluoroaceticacid. After depositing on the analytical plate, the samples were dried.Mass spectrometry analyses were carried out using a MALDI-TOFVoyager-DE-STR type apparatus (Applied BioSystems, Foster City,.Calif.). Deposits containing peptides of less than 3000 Da were analyzedusing the following parameters: positive and reflector modes,acceleration voltage 20 kV, screen tension 61%, delayed extraction 90ns, and mass threshold less than 500 Da. For peptides of 3000 to 10000Da, the parameters were: positive and reflector modes, accelerationvoltage 25 kV, screen tension 65%, delayed extraction 250 ns, and massthreshold less than 1000 Da. The spectra were calibrated externally frommonoisotopic ions [M+H⁺] of different peptides.

For proteins, a 0.5 μl sample was mixed with sinapinic acidextemporaneously dissolved in an amount of 10 mg/ml in a solutioncontaining 50% CH₃CN and 0.1% trifluoroacetic acid. After deposition anddrying, mass spectrometry analyses were carried out using the followingparameters: positive and linear modes, acceleration voltage 25 kV, gridtension 92%, delayed extraction 750 ns, and mass threshold less than1000 Da. The spectra were calibrated externally from the mean masses ofions [M+H⁺] of the thioredoxin of E. coli and of equine apomyoglobin(Applied BioSystems).

I-4—Digestion of Proteins by Endo-Glu and Peptide Separation

1 nanomole of lyophilized HBHA or recombinant HBHA purified bychromatography on heparin-sepharose followed by reverse phase HPLC wasdigested overnight in the presence of 5% Endo-Glu (Roche) in 100 nM ofphosphate buffer (pH 8.0). After enzymatic digestion, the resultingpeptides were separated by reverse phase HPLC using a BeckmanUltrasphere ODS type column (2×200 mm) in a linear elution gradient of 0to 60% acetonitrile prepared in 0.1% trifluoroacetic acid.

I-5—Analysis of Amino Acids and Sequence Determination

To analyze the complete composition of amino acids, native HBHA purifiedby HPLC was hydrolyzed by heating constantly at 110° C. in a 6N HClsolution for 14 to 16 h. The amino acid composition was determined usinga Beckman Gold System type analyzer. The amino-terminal peptide sequencewas determined using the automated Edman degradation method using apulsed liquid apparatus (Procise 492, Applied BioSystems) equipped witha 120 A amino acid analyzer. For each step in the sequencedetermination, the samples comprised 10 to 20 μl, which corresponded toa quantity of peptide of 250 to 500 picomoles.

I-6—Chemical Methylation of Lysine Residues

The method for chemical methylation of recombinant HBHA lysine residueswas derived from the literature (Means, 1977, supra). In substance,recombinant HBHA purified on a heparin-sepharose column was dialyzed for1 h at 4° C. against 250 volumes of 100 mM borate buffer (pH 9.0). Afterdialyis, 3 ml samples of 1 mg/ml protein solution were transferred intoclosed glass tubes containing 70 μl of a freshly prepared solution of 40mg/ml NaBH₄ and 6 μl of 37% formaldehyde solution (formalin, Sigma, StLouis). The tubes were kept in ice. 200 μl samples were removed everyten minutes to verify the degree of completion of the methylationreaction by immunoblotting and mass spectrometry.

I-7—Enzymatic Methylation Test for Recombinant HBHA

100 ml of M. smegmatis or M. bovis BCG cultures with an optical densitymeasured at 600 nm (OD₆₀₀) of 0.5 were centrifuged at 10000 g for 15min. The pellet was re-suspended in 10 ml of 50 mM Hepes buffer (pH 7.4)containing 1 mM of AEBSF (Pefabloc Sc, Roche) and 15% (v/v) of glycerol(buffer A). The cells then underwent continuous sonication for 10minutes at 4° C. using a Branson type sonicator, the outlet power beingadjusted to 5. The total cell lysate was centrifuged at 4° C. at 20000 gfor 15 min. For the methylation tests, 300 μl of total clarified lysatecontaining 1 mg of protein per ml was mixed with 40 μl of[methyl-¹⁴C]AdoMet (60 mCi/mmol, Amersham Pharmacia Biotech), 100 μl ofrecombinant HBHA purified on a heparin column to 0.5 mg/ml, 5 μl of 1MMgCl₂ and 55 μl of buffer A. The methylation tests were carried out at25° C. 100 μl samples were removed at intervals to verify the degree ofmethylation of the recombinant HBHA by autoradiography.

I-8—Animals

The studies were carried out on eight week old female BALB/c mice (IffaCredo, France). For infections with M. tuberculosis, the mice weretransferred into a type P3 confinement.

I-9—Immunization

The mice were immunized three times at two week intervals,subcutaneously at the base of the tail, with 5 μg of native HBHA perdose, emulsified or not emulsified in a solution ofdimethyldioctadecylammonium (DDA, 150 μg/dose, Sigma) andmonophosphorylated lipid A (MPL, 25 μg/dose, Sigma). At the moment ofthe first injection, one group of mice had received a subcutaneous BCGinjection (Paris strain, 5×10⁵ CFU). The mice were infected ten weeksafter the first immunization.

The same experiment was carried out, replacing the native HBHA with (i)non methylated recombinant HBHA and (ii) methylated recombinant HBHA inthe doses for immunization.

I-10—Experimental Infections

As soon as the OD₆₀₀ reached 0.5, the M. tuberculosis cultures werewashed once in Sauton medium, suspended in Sauton medium supplementedwith 30% glycerol then divided into aliquots and finally frozen at −80°C. Prior to infection, an aliquot was defrosted, and the number of CFUswas determined. The mice were infected intravenously into the lateralvein of the tail using an inoculum of 10⁵ CFU of M. tuberculosissuspended in phosphate buffer (PBS, pH 7.4) in a final volume of 200 μl.Four mice per group were sacrificed after six weeks. The number ofbacteria was determined in the spleen, liver and lungs of each infectedmouse, spreading dilutions of the ground organs onto 7H11 medium.

The organs of mice vaccinated with BCG were spread onto 7H11 dishescontaining 2 μg/ml of 2-thiophenecarboxylic acid hydrazide to inhibitthe growth of residual BCG. The colonies were counted after incubatingfor two weeks at 37° C. The protective efficacy was expressed as thelog₁₀ of the reduction in number of bacteria present in the organs ofthe immunized mice compared with the relative enumeration of the groupwhich had received the adjuvant alone. The results were obtained fromgroups of four mice.

I-11—Lymphocyte Culture and IFN-γ-Assay

Spleen lymphocytes were purified as described (Andersen et al, 1991Infect Immun 59: 1558-1563). Lymphocytes from four mice per experimentwere cultured in 96 well plates (NUNC) containing 2×10⁵ cells/well in200 μl of RPMI 1640 (Gibco, France) supplemented with 50 μM of2-mercaptoethanol (Merck, Germany), 50 μg/ml of penicillin-streptomycin(Gibco), 1 mM of glutamax (Gibco) and 10% of foetal calf serum (Roche).

5 μg/ml of concanavalin A was used as the positive control for cellviability. Native HBHA was used in a final concentration of 5 μg/ml. Thesupernatants were recovered 72 hours after the start of stimulation inorder to assay the IFN-γ. IFN-γ was detected using a sandwich type ELISAtest. The anti-IFN-γ monoclonal antibodies used were obtained fromR4-6A2 clones (Pharmingen, USA) for capture and SMG1-2 (Pharmingen) fordetection.

II—Results and Examples

II-1—Characterization and Post-Translational Modification of Native HBHA

Mass spectrometry analysis showed that recombinant HBHA has a molecularweight (MW) of 21340, corresponding to the MW deduced from thenucleotide sequence encoding mycobacterial HBHA (hbhA gene or Rv0475 inM. tuberculosis H37Rv) (Menozzi et al, 1998, supra). In contrast, the MWfor native HBHA was 21610, i.e. 270 more than recombinant HBHA. Inconsequence, the HBHA produced by the mycobacteria underwent amodification, which was not found in the recombinant protein produced byE. coli. In order to define the exact nature of this modification,native and recombinant HBHA underwent hydrolysis with Endo-Glu and themass of the peptides obtained was determined by mass spectrometry. Theonly difference between native and recombinant HBHA was identified atthe carboxy-terminal region of said proteins. The mass of this regionwas 4342 for native HBHA and only 4076 for recombinant HBHA. Thisdifference of about 270 Da corresponded to the mass difference measuredbetween the entire HBHA proteins. Further, the post-translationalmodification or modifications to native HBHA could be localized to theC-terminal region. Further still, the mass spectrum corresponding tothat region was constituted by a single peak for recombinant HBHA, whilefive peaks were present for native HBHA, those peaks being separatedfrom each other by 14 Da (FIG. 1).

II-2—Determination of Post-Translational Modification of Native HBHA

For accurate identification of the modified amino acids, the sequencefor the heparin-binding region was determined using the Edmandegradation method in accordance with conventional procedures. Thisstudy revealed that only the lysines had been modified. Further, of thefifteen lysine residues present in the C-terminal region of HBHA, onlytwo had the standard retention time for lysine. The thirteen otherresidues had retention times corresponding to glutamine and/or argininestandards. Initially, since (i) mass spectrometry analysis showed thatthere was an increment of 14 Da between the different fragments ofnative HBHA, and (ii) only the lysines had been modified, it washypothesized that the lysines in the C-terminal region could have beenmethylated, giving mono-, di- or tri-methyllysines. This hypothesisproved to be only partially accurate, however, as no tri-methyllysinehad been positively identified in the native HBHA. This verification wasmade using standard calibration methods corresponding to mono-, di- andtri-methyllysines respectively. The modified lysines had retention timesthat conformed with those for mono- and di-methyllysine but nottri-methyllysine (FIG. 2).

An amino acid analysis, including the mono-, di- and tri-methyllysine asstandards, confirmed this result.

II-3—Chemical Methylation of Recombinant HBHA

Recombinant HBHA was chemically methylated and then underwent massspectrometrical analysis. As shown in FIG. 3, the mass of the peptidecorresponding to the C-terminal region of the recombinant HBHA increasedas the chemical methylation advanced.

Further, the degree of methylation influenced the reactivity of thepeptides with the monoclonal antibodies 3921E4 and 4057D2 (Rouse et al,1991, supra) (FIG. 4). As described previously (Menozzi et al, 1998,supra), recombinant HBHA was not recognized by antibody 4057D2, althoughit was weakly recognized by antibody 3921E4. In contrast, as shown inFIG. 4, the degree of methylation of the recombinant HBHA affected itsaffinity for these two antibodies in different manners, showing thatmethylation of a protein could play an important role in itsantigenicity.

II-4—Enzymatic Methylation of Recombinant HBHA

In order to determine whether methylation of the lysines of native HBHAwas due to enzymatic activity, an in vitro methylation test specific forrecombinant HBHA was carried out using a mycobacterial lysate.Mycobacterial cultures were lysed by sonication. The total lysates, aswell as the cytoplasmic and parietal fractions were used as enzymaticsources to attempt to transfer [¹⁴C]methyl groups from the[¹⁴C-methyl]AdoMet donor to the acceptor represented by the recombinantHBHA. Incubation of total lysates of M. tuberculosis, M. bovis BCG andM. smegmatis containing [¹⁴C-methyl]AdoMet with recombinant HBHAresulted in the incorporation of [¹⁴C]methyl groups into said HBHA (FIG.2). In contrast, when the lysates were heated to 95° C., they were nolonger capable of catalyzing the transmethylation reaction. Further, themycobacterial methyltransferase or methyltransferases responsible formethylating the HBHA were thermosensitive.

Isolation of the methyltransferase or methyltransferases was envisagedthrough different approaches.

In a first case, the proteins present in a mycobacterial lysate wereseparated by ion exchange chromatography, HPLC or affinity, depending onthe fractions capable of catalyzing the transmethylation reaction from[¹⁴C-methyl]AdoMet onto recombinant HBHA. Such concentration procedurewas continued until a sample was obtained in which the methyltransferaseor methyltransferases were sufficiently pure to determine its sequence.Then, referring to the known sequence of the genome of M. tuberculosisH37Rv (Cole et al, 1998, supra), the gene or genes encoding themethyltransferase or methyltransferases were identified then clonedusing techniques known to the skilled person.

A second approach consisted of seeking candidate genes potentiallyencoding methyltransferases in the genome of M tuberculosis H37Rv on thebasis of sequence homology with the known and identified sequence formethyltransferase genes per se in databases. Five candidate genes wereselected, namely Rv0208c, Rv0380, Rv1405, Rv1644 and Rv3579. These geneswere cloned and expressed in E. coli. The products of said genes werethen purified and tested for their capacity to methylate recombinantHBHA from a radioactively labeled methyl AdoMet donor.

II-5—Production of HBHA by M. smegmatis

It has been demonstrated that M. smegmatis does not express HBHA (Petheet al, 2001, supra). However, it was possible to transfer [¹⁴C]methylgroups from [1⁴C-methyl]AdoMet to recombinant HBHA using a lysate ofthis microorganism (FIG. 2). It was also suggested that M. smegmatis hadthe enzymatic machinery responsible for the HBHA transmethylationreaction. With the aim of verifying this hypothesis, the M. smegmatisMC²155 strain was transformed with a derivative of plasmid pRR3containing the hbhA gene (Rv0475) encoding HBHA in M. bovis BCG, toobtain the M. smegmatis (pRR-hbhA) strain. The production of HBHA wasanalyzed by Western blot. The HBHA produced by M. smegmatis (pRR-hbhA),termed MS-HBHA, was recognized by the monoclonal antibodies 3921E4 and4057D2, strongly suggesting that this MS-HBHA had beenpost-translationally modified, like native HBHA from M. bovis BCG. TheMS-HBHA was purified and underwent hydrolysis by Endo-Glu. Massspectrometry analysis of the digested products thus obtained and peptidesequence determination of the C-terminal region of the MS-HBHA showedthat it effectively had the same type of post-translational modificationas the HBHA from M. bovis. As a consequence, M. smegmatis had anenzymatic machinery that was capable of catalyzing the methylation ofrecombinant HBHA.

As a result, to carry out the vaccination experiments, native HBHA wasalternatively purified from the M. smegmatis transformed strain(pRR-hbhA).

II-6—Study of Native HBHA as a Protective Antigen

The immune response generated by native HBHA, and its protective poweragainst infection by M. tuberculosis, were tested in the murine model.

These experiments were also carried out using recombinant HBHA in thenon methylated and methylated forms.

The immunization protocol was derived from the literature (Brandt et al,2000, Infect Immun 68: 791-795). The adjuvants DDA and MPL were used inamounts of 150 μg and 25 μg per dose respectively.

Group 1 was vaccinated with the adjuvant alone contained in 200 μl ofPBS buffer. Group 2 was vaccinated: with 5 μg of purified native HBHAemulsified in 200 μl of a PBS-adjuvant mixture. Group 3 was vaccinatedwith 5 μg of native HBHA alone in solution in 200 μl of PBS. The micereceived three injections of different preparations at two weekintervals. A fourth group (positive control) was vaccinated with a doseof 5×10⁵ CFU of BCG.

Blood was sampled from all of the mice of the different groups ten daysafter the last injection of the vaccine preparations to test theproduction of antibodies specific to native HBHA. For each group, IgGassays were carried out on serum mixtures. The antibody titer wasdefined as corresponding to the maximum dilution of serums giving avalue three times higher than the blank. Table 1 below shows a readingof the IgG titers induced per injection of the different preparations.TABLE 1 Group 2 Group 1 HBHA + Group 3 Group 4 adjuvant adjuvant HBHABCG total IgG <10 73000 5000 <50 IgG1 <10 580000 24000 <10 IgG2a <1017000 800 <20 IgG2b <10 8500 90 <10 IgG3 <10 750 150 <10

The results show that the mice vaccinated with HBHA (groups 2 and 3)produced large quantities of IgG1and also produced IgG2a, IgG2b andIgG3. These types of antibodies reflect the generation of a mixedTH1/TH2 response. The presence of the adjuvant (group 2) did not modifythe response profile with respect to the HBHA protein alone (group 3).However, said adjuvant could produce about 10 times more of thedifferent IgGs (Table 1).

To test the cell response, four mice per group were sacrificed ten weeksafter the first injection. The lymphocytes were collected and stimulatedin vitro with native HBHA. After stimulation, the IFN-γ production wastested. As shown in FIG. 5, only the lymphocytes purified from mice ofgroup 2, vaccinated with native HBHA associated with adjuvant, producedIFN-γ specific to said HBHA.

Finally, experiments were carried out with the aim of testing theprotective power of native HBHA against an infection with M.tuberculosis. Group 3, vaccinated with HBHA alone, was set aside infavor of group 2, given that the immune response, both humoral (Table 1)and cellular (FIG. 5) appeared to be of better quality in group 2 in thelight of the experimental results. Ten weeks after the first injectionof the vaccine preparations, mice were intravenously infected with 10⁵CFU of M. tuberculosis. Four mice per group were sacrificed six weeksafter infection to determine the number of CFUs present in the differentmouse organs. The bacterial charge was determined in the liver, spleenand lungs of the animals. Resistance was defined as the difference inbacterial charge, expressed as the log₁₀, between the control group 1,vaccinated with adjuvant alone, and groups 2 and 4, respectivelyvaccinated with HBHA associated with adjuvant and with BCG. Table 2below shows the efficacy of the protection induced by the differentimmunizations. TABLE 2 Liver Spleen Lungs CFU CFU CFU (log₁₀) Resistance(log₁₀) Resistance (log₁₀) Resistance Group 1 5.60 ± 0.20 5.85 ± 0.215.27 ± 0.25 adjuvant Group 2 4.66 ± 0.35 0.94 5.00 ± 0.04 0.85 4.34 ±0.17 0.93 HBHA + adjuvant Group 4 4.41 ± 0.20 1.19 4.68 ± 0.25 1.17 4.45± 0.20 0.82 BCG

Determining the CFUs showed that the immune response caused by nativeHBHA was capable of rendering the mouse partially resistant to infectionby M. tuberculosis. The observed resistance was of the same order ofmagnitude, both for the native HBHA and the prior art reference vaccine,namely BCG. As a result, injection of native HBHA would protect themouse from infection with M. tuberculosis, in proportions close to thosefor the BCG vaccine.

This experiment was carried out using the methylated and non methylatedforms of the recombinant HBHA to compare the level and efficacy of theinduced protection with that observed with native HBHA. Thus, methylatedrecombinant HBHA, in that it is immunogenic, causes resistance inanimals to an infection with M. tuberculosis that is as effective asthat induced by native HBHA.

In a further aspect, the present invention provides a sub-unit vaccineintended for the treatment of mycobacterial infections and comprisingnative HBHA in its formulation.

Within the context of the production of vaccine compositions on anindustrial scale, it is politic to use genetically recombinantproducting organisms which are often more advantageous than wildproducting organisms in that the former can easily be transformed by thenucleotide sequences of the latter, encoding the protein or proteins ofinterest, and in that they are carefully selected, in particular fortheir harmlessness and their readily controllable growth parameters, andso there is no need to invest in expensive specialized equipment. Forthis reason, a preferred aspect of the invention concerns a sub-unitvaccine for the treatment of mycobacterial infections advantageouslycharacterized in that it comprises in its formulation methylated HBHA inits recombinant version, i.e. produced by a recombinant host cellmeticulously selected to satisfy industrial and safety requirements.

1. An immunogenic recombinant peptide sequence, characterized in that itis a methylated form of an expression product of a nucleotide sequenceencoding an HBHA type mycobacterial antigen, in particular an antigenobtained from M. bovis BCG or M. tuberculosis.
 2. An immunogenicrecombinant peptide sequence according to claim 1, characterized in thatthe nucleotide sequence encodes the heparin-binding region of the HBHA.3. An immunogenic recombinant peptide sequence according to claim 1 orclaim 2, characterized in that the methyl groups are carried by lysineresidues located in the heparin-binding region of the HBHA.
 4. Animmunogenic recombinant peptide sequence according to claim 3,characterized in that the lysine residues are mono- or di-methylated. 5.An immunogenic recombinant peptide sequence according to claim 3 orclaim 4, characterized in that the methyl groups are carried by all or aportion of the lysine residues located in the heparin-binding region ofthe HBHA.
 6. An immunogenic recombinant peptide sequence according toany one of claims 3 to 5, characterized in that the methyl groups arecarried by all of the lysine residues located in the heparin-bindingregion of the HBHA.
 7. An immunogenic recombinant peptide sequenceaccording to any one of claims 1 to 6, characterized in that it isrecognized by the monoclonal antibody 4057D2.
 8. A method for producingan immunogenic recombinant peptide sequence according to any one ofclaims 1 to 7, characterized in that it comprises at least the followingsteps: producing recombinant HBHA protein using a recombinant host cell;purifying said protein; post-translational methylation thereof; theorder of the last two steps optionally being reversed.
 9. A productionmethod according to claim 8, characterized in that the recombinant HBHAprotein is constituted by its heparin-binding region.
 10. A productionmethod according to claim 8 or claim 9, characterized in that therecombinant host cell is a bacterium.
 11. A production method accordingto claim 10, characterized in that the bacterium is M. smegmatis.
 12. Aproduction method according to any one of claims 8 to 11, characterizedin that the methylation step is carried out chemically.
 13. A productionmethod according to any one of claims 8 to 11, characterized in that themethylation step is carried out enzymatically.
 14. A production methodaccording to claim 13, characterized in that the methylation reaction iscatalyzed by at least one methyltransferase contained in extracts fromtotal mycobacterial proteins, in particular M. bovis BCG or M.smegmatis.
 15. A production method according to claim 13 or claim 14,characterized in that the methylation reaction is catalyzed by at leastone purified mycobacterial methyltransferase.
 16. A production methodaccording to claim 13 or claim 15, characterized in that the recombinanthost cell co-produces HBHA and the recombinant methyltransferase ormethyltransferases.
 17. A production method according to claim 16,characterized in that each recombinant protein is encoded by anexpression vector.
 18. A production method according to claim 16,characterized in that all or a portion of the recombinant proteinsis/are encoded by the same expression vector.
 19. A recombinant vector,characterized in that it encodes HBHA and at least one recombinantmycobacterial methyltransferase.
 20. A recombinant vector according toclaim 19, characterized in that production of each recombinant proteinis controlled by distinct regulation sequences.
 21. A recombinant vectoraccording to claim 19, characterized in that production of all or partof the recombinant proteins is controlled by the same regulationsequences.
 22. A recombinant host cell holding a recombinant vectoraccording to any one of claims 19 to
 21. 23. A method for producing animmunogenic recombinant peptide sequence according to any one of claims1 to 7, characterized in that it comprises at least the following steps:a) co-producing the HBHA protein and the mycobacterial methyltransferaseor methyltransferases by a host cell according to claim 22; b)post-translational methylation of the recombinant HBHA by therecombinant methyltransferase or methyltransferases; and c) purifyingthe methylated recombinant HBHA.
 24. Methylated immunogenic recombinantpeptide sequences that can be obtained using a method according to anyone of claims 8 to 18 and
 23. 25. Use of an immunogenic recombinantpeptide sequence according to any one of claims 1 to 7, to preparevaccines against mycobacterial infections, in particular infections withM. bovis or M. tuberculosis.
 26. An immunogenic composition,characterized in that it comprises, as an active principle, a methylatedform of HBHA in a pharmaceutically acceptable formulation.
 27. Animmunogenic composition according to claim 26, characterized in that themethylated form is associated with one or more adjuvants.
 28. Animmunogenic composition according to claim 26 or claim 27, characterizedin that the methylated form is native HBHA.
 29. An immunogeniccomposition according to claim 26 or claim 27, characterized in that themethylated form is a recombinant peptide sequence according to any oneof claims 1 to
 7. 30. An immunogenic composition according to claim 28or claim 29, characterized in that it comprises between 0.1 and 20 μg ofpurified HBHA protein per dose.
 31. An immunogenic composition accordingto claim 30, characterized in that it comprises 5 μg of purified HBHAprotein per dose.